Effects Of Yeast Extract On Carbon And Nitrogen Metabolism And Metabolism Of Tropane Alkaloids In Hairy Roots Of Atropa Belladonna L.

Atropa belladonna L.is an important perennial herb in the Atropa of the Solanaceae.The major secondary metabolites hyoscyamine and scopolamine are widely used in clinical practice for antispasmodic,analgesic,antiemetic,anesthesia and treatment of Parkinson’s disease etc.As the extremely low content of hyoscyamine and scopolamine in Atropa belladonna L.,especially the content of scopolamine is far less than hyoscyamine,the yield of these two tropane alkaloids are far below the market demand.Therefore,how to improve the secondary metabolites has became the key research direction of Atropa belladonna L.And using hairy roots to get more secondary metabolites by adding different kind of elicitors is a hot research field of Atropa belladonna L.recently.In this study,Atropa belladonna L.hairy roots were treated with yeast extract（YE）and YE was added to the culture medium.Then the changes of physiological and biochemical indexes of Atropa belladonna L.hairy roots after treatment with YE were detected.Including some indexes of carbon metabolism,nitrogen metabolism and antioxidant metabolism.Furthermore,the content of amino acid,polyamine,the relative expression of key enzyme genes and the yield of major metabolites hyoscyamine and scopolamine in tropane alkaoid metabolic pathway were measured to analysis the mechanism that how YE effect the growth and metabolism of Atropa belladonna L.hairy roots,and how to induced the synthesis of secondary metabolites.The results are as follows.1.Compared the effects of different concentrations of YE on hyoscyamine and scopolamine yield of Atropa belladonna L.hairy roots,the results showed that when the concentration of YE was 100 mg·L^-1,the yield of alkaloids could be efficiently accumulated in a short period of time.Adding a higher YE concentration,such as 300mg·L^-1,could reduce the yield of hyoscyamine and scopolamine.When the concentration of YE was 400 mg·L^-1,it even caused the death of Atropa belladonna L.hairy roots.2.Studied the effects of YE on carbon metabolism of Atropa belladonna L.hairy roots,the results showed that the carbon metabolism of the Atropa belladonna L.hairy roots in the control group（CK）was in a stable state throughout the entire culture process.But the addition of YE changed the original adjusted method of carbon metabolism of Atropa belladonna L.hairy roots.YE caused the whole carbon metabolism more active mainly by enhancing the activity of amylase,then the total carbon content increased significantly,while the soluble sugar content did not change significantly.3.Studied the effects of YE on nitrogen metabolism of Atropa belladonna L.hairy roots,the results showed that the activity of key enzymes of nitrogen metabolism changed differently.Compared with the CK,the activity of nitrate reductase（NR）and glutamine synthetase（GS）were significantly increased,while the activity of glutamate dehydrogenase（GDH）was not changed significantly.The content of nitrate nitrogen,ammonium nitrogen had a significant decrease,but the content of soluble protein,free amino acid,total nitrogen were significantly more than CK.It can be seen that YE accelerated nitrogen metabolism of Atropa belladonna L.hairy roots and enhanced nitrogen assimilation.4.Studied the effects of YE on anti-oxidation metabolism of Atropa belladonna L.hairy roots,the results showed that the active oxygen accumulated continuously in the culture time.Atropa belladonna L.hairy roots can remove active oxygen by enhancing the activity of various antioxidant enzymes to maintain normal growth and metabolism.Compared with CK,the addition of YE increased the activity of catalase（CAT）and superoxide dismutase（SOD）,and the activity of peroxidase（POD）decreased significantly after a period of treatment,which indicated that the active oxygen content in hairy roots decreased,avoiding the damage of excess active oxygen to cells.5.Studied the effects of YE on tropane alkaloid metabolism of Atropa belladonna L.hairy roots,the results showed that YE treatment led to the increase of the content of precursor amino acids（Ornithine and arginine）and precursor putrescine in secondary metabolic pathways of Atropa belladonna L.hairy roots.The expression level of gene putrescine N-methyl transferase（PMT）,tropinone reductase-I（TR I）and hyoscyamine6-β-hydroxylase（H6H）all increased in a different rate caused by YE treatment.And eventually led to the increase of the yield of tropane alkaloids.The yield of hyoscyamine and scopolamine were 3.09 and 1.85 folds than CK after 16 days treatment time.In summary,YE has a certain effect on the nitrogen metabolism and antioxidant metabolism of Atropa belladonna L.,but it effected hairy roots mainly by enhancing the nitrogen metabolism.YE induced more synthesis of tropane alkaloids by increasing the activity of key enzymes in nitrogen metabolism to provide more synthetic materials for secondary metabolism.Meanwhile it regulated the expression level of some genes of key metabolic enzyme to accelerate secondary metabolism and led to the increase of the yield of hyoscyamine and scopolamine.This study can provide a good reference for the increase of the effective medicinal components of Atropa belladonna L.in actual production,and also provide some theoretical support for the tropane alkaloids metabolic engineering.